Project Summary Eosinophilic esophagitis (EoE) is an increasingly prevalent chronic inflammatory disease of the esophagus, mediated by dietary food antigens and clinically characterized by upper gastrointestinal (GI) symptoms including dysphagia and food impaction. Recently, a confounding esophageal disorder, termed proton-pump inhibitor (PPI)?responsive esophageal eosinophilia (PPI-REE), has emerged; PPI-REE is indistinguishable from EoE by clinical, endoscopic or histologic features or by gene profiles. The current clinical conundrums are whether PPI- REE represents a GERD-related phenomenon, a subtype of EoE or a completely new entity and why PPI-REE and EoE respond differently to PPI. RNA sequencing (RNA-Seq) analyses of esophageal biopsy samples from patients with active EoE disease revealed dysregulation of gene networks associated with regulating intracellular [pH]i and acid protection and that the most upregulated transmembrane transporter activity gene was SLC9A3, which encodes for the sodium-hydrogen exchanger family member 3 (NHE3). Recently, we have demonstrated 1) increased expression of SLC9A3 within the basal layer of ESSE biopsies from patients with EoE and that expression positively correlated with disease severity (eosinophils/HPF) and DIS; 2) IL-13 induced SLC9A3 expression and function in ESSE cells and that SLC9A3 activity positively correlating with DIS formation and 3) SLC9A3-mediated Na+-dependent proton secretion is the primary intracellular acid protective mechanism within IL-13?stimulated ESSE cells and blockade of this pathway abrogated DIS formation45. In new preliminary studies we have made several transformative observations: 1) IL-13 induced expression of the transcription factor aryl hydrocarbon receptor (AhR) and AhR-responsive genes in ESSE cells and EoE biopsies; 2) stimulating ESSE cells with AhR ligands, suppressed AhR-responsive gene expression including SLC9A3 and 3) a divergent effect of PPI therapy on SLC9A3 expression in ESSE biopsy samples from individuals with EoE and PPI-REE, suggesting an opposing impact of PPI on SLC9A3 transcriptional regulation between EoE and PPI-REE. Collectively, these observations underlie our central hypothesis that SLC9A3 activity promotes DIS formation in EoE subtypes and that this pathway is divergently responsive to PPI therapy via AhR-dependent signaling. The specific Aims outlined in this proposal will 1) Aim 1. Determine the relationship between SLC9A3 expression and function, disease severity and DIS formation in EE subtypes; 2) Define the requirement of SLC9A3 in ESSE DIS formation and 3) Define the involvement of PPI-induced AhR signaling in Type-2 cytokine-induced SLC9A3 expression and function in ESSE cells. With respect to the expected outcomes, the studies proposed in Aim I are expected to establish the contribution of SLC9A3 to histopathologic features of EoE and PPI-REE and responsiveness of this pathway to PPI trial; Aim II are expected to determine the necessity of SLC9A3 to ESSE acid transport and DIS and Aim III is expected to determine the interaction between IL-13? and PPI-induced AhR signaling in SLC9A3 expression and function in ESSE cells and DIS formation. Successfully completing the proposed studies will provide a new and substantive departure from our current understanding of the underlying molecular mechanisms underpinning the development of PPI-REE and EoE and provide an explanation for their differential responsiveness to PPI therapy, thereby directing the development of new and pre-existing therapeutics for treating esophageal eosinophilia?related disorders.